The present invention relates to a serum-free medium for the growth and maintenance of mammalian cells in culture.
Cell culture is widely used today for the production of various biologically active products, such as viral vaccines, monoclonal antibodies, non-antibody immuno-regulators, polypeptide growth factors, hormones, enzymes, tumor specific antigens, etc. These products are produced by normal or transformed and genetically engineered cells.
For culturing cells, in the past the culture medium was supplemented with serum, which serves as a universal nutrient for the growth and maintenance of all mammalian cell lines that produce biologically active products. Serum contains hormones, growth factors, carrier proteins, attachment and spreading factors, nutrients, trace elements, etc. Culture media usually contained up to about 10% of animal serum, such as fetal bovine serum (FBS), also called fetal calf serum (FCS).
Although widely used, serum has many limitations. It contains high levels of numerous proteins interfering with the limited quantities of the desired protein of interest produced by the cells. These proteins derived from the serum must be separated from the product during downstream processing such as purification of the protein of interest, which complicates the process and increases the cost.
The advent of BSE (Bovine Spongiform Encephalopathy), a transmissible neurodegenerative disease of cattle with a long latency or incubation period, has raised regulatory concerns about using animal-derived sera in the production of biologically active products.
There is therefore a great demand for the development of alternative media free from animal serum that support cell growth and maintain cells during the production of biologically active products.
Generally, cell culture media comprise many components of different categories, such as amino acids, vitamins, salts, fatty acids, and further compounds:                Amino acids: For instance, U.S. Pat. No. 6,048,728 (Inlow et al.) discloses that the following amino acids may be used in a cell culture medium: Alanine, Arginine, Aspartic Acid, Cysteine, Glutamic Acid, Glutamin, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenyalanine, Proline, Serine, Tryptophan, Tyrosine, Threonine, and Valine.        Vitamins: US 2003/0096414 (Ciccarone et al.) or U.S. Pat. No. 5,811,299 (Renner et al.) for example describe that the following vitamins may be used in a cell culture medium: Biotin, Pantothenate, Choline Chloride, Folic Acid, Myo-Inositol, Niacinamide, Pyridoxine, Riboflavin, Vitamin B12, Thiamine, Putrescine.        Salts: For instance, U.S. Pat. No. 6,399,381 (Blum et al.) discloses a medium comprising CaCl2, KCl, MgCl2, NaCl, Sodium Phosphate Monobasic, Sodium Phosphate Dibasic, Sodium Selenite, CuSO4, ZnCl2. Another example for a document disclosing the inorganic salts that may be used in a culture medium is US 2003/0153042 (Arnold et al.), describing a medium comprising CaCl2, KCl, MgCl2, NaCl, Sodium Phosphate Monobasic, Sodium Phosphate Dibasic, CuCl2.2H2O, ZnCl2.        Fatty acids: Fatty acids that are known to be used in media are Arachidonic Acid, Linoleic Acid, Oleic Acid, Laurie Acid, Myristic Acid, as well as Methyl-beta-Cyclodextrin, see e.g. U.S. Pat. No. 5,045,468 (Darfler). It should be noted that cyclodextrin is not a lipid per se, but has the ability to form a complex with lipids and is thus used to solubilize lipids in the cell culture medium.        Further components, in particular used in the frame of serum-free cell culture media, are compounds such as glucose, glutamine, Na-pyruvate, insulin or ethanolamine (e.g. EP 274 445), or a protective agent such as Pluronic F68. Pluronic® F68 (also known as Poloxamer 188) is a block copolymer of ethylene oxide (EO) and propylene oxide (PO).        
Standard “basic media” are also known to the person skilled in the art. These media already contain several of the medium components mentioned above. Examples of such media that are widely applied are Dulbecco's Modified Eagle's Medium (DMEM), DMEM F12 (1:1), Ham's Nutrient mixture F-10, Roswell Park Memorial Institute Medium (RPMI), MCDB 131, or William's Medium E. These commercial media are available e.g. from Gibco, Invitrogen.
Metals such as Zinc (Zn) and Copper (Cu) are involved in metabolic reactions (Vallee and Falchuk, 1993, or Lindner, 1991).
Zinc is essential to the structure and function of a large number of macromolecules and for many enzymatic reactions. It plays a catalytic, co-catalytic or structural role in the proper folding of proteins. Zn-ATP is necessary for the synthesis of pyridoxal-5-phosphate and flavin adenosine dinucleotide (FAD), two coenzymes essential for biogenic amine synthesis and monoamine oxidase metabolism.
The activity of Zinc in protecting biological structures from damage by free radicals may be due to several factors: maintaining the adequate level of metalloproteins, which are also free radical scavengers, as an essential component of superoxide dismutase, as a protective agent for thiols, and in preventing the interaction of chemical groups with iron to form free radicals.
In addition to that, the presence of Zn prevents lipid peroxidation. Zinc is also an effector of tubulin polymerization and acts in vitro on actin filament formation and stabilization. Zinc is also a component of the Zinc finger motif of DNA binding proteins, which is a common motif in transcription proteins.
Zinc ions exist primarily in the form of complexes with proteins and nucleic acids and participate in all aspects of intermediary metabolism, transmission and regulation of the expression of genetic information, storage, synthesis and action of peptide hormones and structural maintenance of chromatin and bio-membranes.
Copper is also a trace element important for the function of many cellular enzymes. Copper ions can adopt distinct redox states, oxidized Cu (II), or reduced Cu (I), allowing the metal to play a pivotal role in cell physiology as a catalytic cofactor in the redox-chemistry of enzymes. It functions in a group of copper oxidases, which include cytochrome c oxidase, tyrosinase, dopamine-β-monooxygenase, amine oxidases and lysyl oxidase. Copper also participates in mitochrondrial respiration, iron homeostasis as a component of ceruloplasmin, free radical scavenging and elastin cross-linking.
Serum-free media comprising metal ions such as zinc or copper ions are known in the art, e.g. from U.S. Pat. No. 6,048,728 (Inlow et al.), U.S. Pat. No. 4,767,704 (Cleveland et al.) or WO 01/16294 (Life Technologies Inc.). However, these documents do not describe a productivity-enhancing effect of these ions if added at specific concentrations to a standard production medium.
For the development and supply of biologically active products, such as therapeutic proteins or vaccines, large amounts must be produced. Suitable cells that are widely used for production of polypeptides are Chinese Hamster Ovary (CHO) cells. CHO cells were first cultured by Puck (J. Exp. Med. 108, 945, 1958) from a biopsy of an ovary from a female Chinese hamster. From these original cells a number of sub-lines were prepared with various characteristics. One of these CHO cell lines, CHO-K1, is proline-requiring and is diploid for the dihydrofolate reductase (DHFR) gene. Another line derived from this cell line is a DHFR deficient CHO cell line (CHO DUK B11) (PNAS 77, 1980, 4216-4220), which is characterized by the loss of DHFR function as a consequence of a mutation in one DHFR gene and the subsequent loss of the other gene.
Further cells that are frequently used for the production of proteins intended for administration to humans are human cell lines such as the human fibrosarcoma cell line HT1080 or the human embryonic kidney cell line 293.
The murine C127 cell line is also highly suitable for production of recombinant proteins (Carter et al., 1989; Oka and Rupp, 1990).
One therapeutic protein of interest is growth hormone. Human growth hormone (hGH), also known as somatropin (INN) or somatotropin, is a protein hormone produced and secreted by the somatotropic cells of the anterior pituitary. Human growth hormone plays a key role in somatic growth in childhood and in metabolism in adulthood through its effects on the metabolism of proteins, carbohydrates and lipids.
Human growth hormone is a single polypeptide chain of 191 amino acids (Bewly et al, 1972) having two disulfide bonds, one between Cys-53 and Cys-165, forming a large loop in the molecule, and the other between Cys-182 and Cys-189, forming a small loop near the C-terminus. The DNA sequence that confirmed the amino acid sequence was reported by Martial et al (1979). Purified hGH is a white amorphous powder in its lyophilized form. It is readily soluble (concentrations>10 mg/L) in aqueous buffers at pH in a range of 6.5 to 8.5.
In solution, hGH exists predominantly as a monomer, with a small fraction as dimers and higher molecular weight oligomers. Under certain conditions, hGH can be induced to form larger amounts of dimers, trimers and higher oligomers.
Several derivatives of hGH are known, including naturally-occurring derivatives, variants and metabolic products, degradation products primarily of biosynthetic hGH and engineered derivatives of hGH produced by genetic methods. One example of a naturally-occurring derivative of hGH is GH-V, a variant of growth hormone found in the placenta. Other members of the gene locus are described in Chen et al (1989). Any derivative of hGH, including derivatives designed to be long-lasting in the body, can be used for the purpose of the present invention as long as it retains the biological activity of hGH.
Methionyl hGH was the first form of hGH to be produced through recombinant DNA technology. This compound is actually a derivative of hGH having one additional methionine residue at its N-terminus (Goeddel et al, 1979).
A naturally-occurring variant of hGH called 20-K-hGH has been reported to occur in the pituitary as well as in the bloodstream (Lewis et al, 1978; Lewis et al, 1980). This compound, which lacks the 15 amino acid residues from Glu-32 to Gln-46, arises from an alternative splicing of the messenger ribonucleic acid (DeNoto et al, 1981). This compound shares many, but not all of the biological properties of hGH.
20-K-hGH is made in the pituitary and secreted into the blood. It makes up about 5% of growth hormone output of adults, and about 20% of growth hormone output of children. It has the same growth promoting activity as 22 kD growth hormone, and has been reported to have equal to or greater than the amount of lipolytic activity as the 22 kD form. It binds to growth hormone receptors with equal affinity as the 22 kD growth hormone, and has one tenth the lactogenic (prolactin-like) bioactivity as the 22 kD hormone. Unlike 22 kD, the 20-k-hGH has weak anti-insulin activity.
A number of derivatives of hGH arise from proteolytic modifications of the molecule. The primary pathway for the metabolism of hGH involves proteolysis. The region of hGH around residues 130-150 is extremely susceptible to proteolysis, and several derivatives of hGH having nicks or deletions in this region have been described (Thorlacius-Ussing, 1987). This region is in the large loop of hGH, and cleavage of a peptide bond there results in the generation of two chains that are connected through the disulfide bond at Cys-53 and Cys-165. Many of these two-chain forms are reported to have increased biological activity (Singh et al, 1974). Many derivatives of human growth hormone have been generated artificially through the use of enzymes. The enzymes trypsin and subtilisin, as well as others, have been used to modify hGH at various points throughout the molecule (Lewis et al, 1977; Graff et al, 1982). One such derivative, called two-chain anabolic protein (2-CAP), was formed through the controlled proteolysis of hGH using trypsin (Becker et al, 1989). 2-CAP was found to have biological properties very distinct from those of the intact hGH molecule, in that the growth-promoting activity of hGH was largely retained and most of the effects on carbohydrate metabolism were abolished.
Asparagine and glutamine residues in proteins are susceptible to deamidation reactions under appropriate conditions. Pituitary hGH has been shown to undergo this type of reaction, resulting in conversion of Asn-152 to aspartic acid and also, to a lesser extent, conversion of Gln-137 to glutamic acid (Lewis et al, 1981). Deamidated hGH has been shown to have an altered susceptibility to proteolysis with the enzyme subtilisin, suggesting that deamidation may have physiological significance in directing proteolytic cleavage of hGH. Biosynthetic hGH is known to degrade under certain storage conditions, resulting in deamidation at a different asparagine (Asn-149). This is the primary site of deamidation, but deamidation at Asn-152 is also seen (Becker et al, 1988). Deamidation at Gln-137 has not been reported in biosynthetic hGH.
Methionine residues in proteins are susceptible to oxidation, primarily to the sulfoxide. Both pituitary-derived and biosynthetic hGH undergo sulfoxidations at Met-14 and Met-125 (Becker et al, 1988). Oxidation at Met-170 has also been reported in pituitary but not biosynthetic hGH. Both desamide hGH and Met-14 sulfoxide hGH have been found to exhibit full biological activity (Becker et al, 1988).
Truncated forms of hGH have been produced, either through the actions of enzymes or by genetic methods. 2-CAP, generated by the controlled actions of trypsin, has the first eight residues at the N-terminus of hGH removed. Other truncated versions of hGH have been produced by modifying the gene prior to expression in a suitable host. The first 13 residues have been removed to yield a derivative having distinctive biological properties (Gertler et al, 1986) in which the polypeptide chain is not cleaved.
Although human growth hormone was originally obtained from pituitary glands of cadavers, these preparations were not electrophoretically homogeneous, and antibodies appeared in the serum of patients treated with preparations of the order of 50% purity, the immunogenicity being attributed to inactive components. Recombinant DNA technology permitted production of an unlimited supply of hGH in a number of different systems. Purification of hGH from the culture medium is facilitated by the presence of only low amounts of contaminating proteins. In fact, it has been shown that hGH can be purified on a laboratory scale by a single purification step on a reversed-phase HPLC column (Hsiung et al (1989).
Recombinant human growth hormone, rhGH, is produced by Serono International S.A. as SEROSTIM®, which product has been given accelerated FDA approval for treating weight loss and wasting in AIDS patients. SAIZEN® is recombinant human growth hormone indicated for GH deficiency in children, for Turner syndrome in girls, as well as chronic renal failure in children. PROTROPIN®, produced by Genentech, Inc. (South San Francisco, Calif.), differs slightly in structure from natural sequence hGH, having an additional methionine residue at the N-terminus. Recombinant hGH is generally marketed as vials containing hGH plus additional excipients, e.g., glycine and mannitol, in a lyophilized form. A companion diluent vial is provided, allowing the patient to reconstitute the product to the desired concentration prior to administration of the dose. Recombinant hGH can also be marketed in other well-known manners, such as prefilled syringes, etc.
In general, no significant differences have been observed in the pharmacokinetics or biological activities of recombinant natural sequence hGH, recombinant N-methionyl-hGH, or pituitary-derived material in humans (Moore et al, 1988; Jorgensson et al, 1988).
In view of the various medical indications for which growth hormone is used, there is a need for an efficient and safe way of producing sufficient quantities of it in cell culture, in particular in a serum-free cell culture process.
Serum-free media have been described in the art.
For instance, U.S. Pat. No. 6,162,643 describes a serum-free basal medium designated HECK-109, containing trace amounts of Copper Sulphate and Zinc Chloride. This medium is specifically designed for primary and secondary cultures of normal human cells such as keratinocytes with the aim of tissue generation for human transplantation. Expression of recombinant human proteins in cell lines in vitro is not mentioned in this US patent.
U.S. Pat. No. 5,324,656 discloses a serum-free basal medium called MCDB120 and MCDB 131M, comprising trace amounts of Copper Sulphate and Zinc Chloride. This medium is specifically designed for the in vitro culture of human muscle satellite cells with the aim of preventing differentiation of these cells. Production of recombinant human proteins is not envisaged in the frame of this document.
GB 2 196 348 describes a synthetic medium for the in vitro culture of hybridoma and myeloma cells. The medium contains Copper, Zinc and Ferric ions. Cultivation of the hybridoma or myeloma cells in this medium is exclusively for the purpose of manufacturing monoclonal antibodies.
U.S. Pat. No. 6,103,529 provides for a serum-free cell culture medium formulation for the in vitro cultivation of animal cells. The animal cells may be used for the production of viruses, monoclonal antibodies, hormones or growth factors. However, the production of growth hormone is not mentioned in this document.
U.S. Pat. No. 6,048,728 discloses a protein-free cell culture medium comprising Co-, Zn- and Fe-Ions for the cultivation of animal cells in order to produce e.g. natural or recombinant products, such as antibodies. Such products to be produced by the cultured cells do not include growth hormones or growth factors, which are exclusively mentioned as constituents of the serum-free medium in order to enhance cell growth of in culture.
U.S. Pat. No. 5,316,938 discloses a biochemically defined culture medium for Chinese Hamster Ovary (CHO) cells comprising Ferric Citrate, Zinc sulphate and Copper sulphate, named WCM5. The medium is specifically designed for antibody and tPA production in CHO cells.
U.S. Pat. No. 5,122,459 describes a method for production of recombinant proteins in a serum-free culture medium containing Zinc and Ferric ions, particularly suitable for the culture of CHO cells. Production of growth hormone is not mentioned in this document.
Therefore, the problem underlying the present invention is providing a serum-free cell culture medium for the efficient production of Growth Hormone, in particular human Growth Hormone (hGH).